Local production of lactate, ribose phosphate, and amino acids within human triple-negative breast cancer

Abstract

Background: Upregulated glucose metabolism is a common feature of tumors. Glucose can be broken down by either glycolysis or the oxidative pentose phosphate pathway (oxPPP). The relative usage within tumors of these catabolic pathways remains unclear. Similarly, the extent to which tumors make biomass precursors from glucose, versus take them up from the circulation, is incompletely defined.

Methods: We explore human triple negative breast cancer (TNBC) metabolism by isotope tracing with [1,2-13C]glucose, a tracer that differentiates glycolytic versus oxPPP catabolism and reveals glucose-driven anabolism. Patients enrolled in clinical trial NCT03457779 and received IV infusion of [1,2-13C]glucose during core biopsy of their primary TNBC. Tumor samples were analyzed for metabolite labeling by liquid chromatography-mass spectrometry (LC-MS). Genomic and proteomic analyses were performed and related to observed metabolic fluxes.

Findings: TNBC ferments glucose to lactate, with glycolysis dominant over the oxPPP. Most ribose phosphate is nevertheless produced by oxPPP. Glucose also feeds amino acid synthesis, including of serine, glycine, aspartate, glutamate, proline and glutamine (but not asparagine). Downstream in glycolysis, tumor pyruvate and lactate labeling exceeds that found in serum, indicating that lactate exchange via monocarboxylic transporters is less prevalent in human TNBC compared with most normal tissues or non-small cell lung cancer.

Conclusions: Glucose directly feeds ribose phosphate, amino acid synthesis, lactate, and the TCA cycle locally within human breast tumors.

Figures

Figure 1.. Infusion of [1,2-13C]glucose in patients with TNBC

a) Infusion of [1,2-13C]glucose. Blue circles represent 13C, red arrows indicate serum collection and black arrow biopsy collection. b) Serum isotopic labeling of glucose, pyruvate and lactate from [1,2-13C]glucose during infusion (mean, ± 95% confidence interval, n = 12 at 30 min, n = 11 at 60 min, 3 technical replicates per patient). c) Average per carbon labeling of circulating lactate normalized to that of circulating glucose in indicated human and mouse infusion studies (mean ± SEM, n = number of individuals infused). See also Figure S1.

Figure 2.. Glycolytic flux exceeds pentose phosphate activity in human breast tumors

a) Tracer strategy for comparing glycolysis and pentose phosphate pathway flux using [1,2-13C]glucose. Blue circles depict glycolytic metabolites produced exclusively by glycolysis (M+2 labeling); orange circles depict glycolytic intermediates arising from glucose that first passed through the oxPPP (M+1). b) Individual patient M+1 and M+2 labeling of tumor lactate (mean ± SEM, n = 2 needle biopsy samples per patient), and c) M+1 and M+2 labeling of tumor glycolytic species (mean ± SEM, n = 24 total biopsy samples collected from twelve patients) from [1,2-13C]glucose. Dominance of M+2 fraction means that lower glycolytic intermediates are mainly from glycolysis, not oxPPP flux. G6P, glucose-6-phosphate; 3PG, 3-phosphoglycerate; R5P, ribose-5-phosphate and its isomers ribulose-5-phosphate and xyulose-5-phosphate. See also Figure S2.

Figure 3.. oxPPP is primary source of ribose phosphate in TNBCs

a) Tracer strategy for comparing oxPPP and non-oxPPP activity using [1,2-13C]glucose. Blue circles depict the path tracer 13C carbons take to produce R5P via the non-oxPPP; orange circles depict the path taken via the oxPPP. Since one glucose carbon is lost as CO2 during oxPPP catabolism, M+1 labeling of R5P indicates oxPPP production, while M+2 labeling indicates non-oxPPP production. b) Individual patient labeling of R5P in TNBCs tumors (mean ± SEM, n = 2 biopsy samples per patient). c) Total labeling (the sum of M+1 and M+2) of R5P correlates with that of G6P. Fit using linear regression. d) Average tumor labeling of G6P and R5P (mean ± SEM, n = 24 total biopsy samples collected from twelve patients). Dominance of M+1 fraction indicates the oxPPP is the primary PPP arm for producing R5P. G6P, glucose-6-phosphate; R5P, ribose-5-phosphate and its isomers ribulose-5-phosphate and xyulose-5-phosphate.

Figure 4.. TNBCs produce serine and glycine from glucose

a) Tracing of de novo serine/ glycine production using [1,2-13C]glucose. Blue circles (M+2 labeling) depict 13C carbons arising directly from [1,2-13C]glucose by de novo serine production; orange circles (M+1 labeling) depict labeled glycine and serine arising from subsequent reversible SHMT activity. b) Individual patient labeling of serine in TNBCs (mean ± SEM, n = 2 biopsy samples per patient). c) Fractional carbon labeling of tumor glycine correlates with that of tumor serine. d) Fractional carbon tumor serine labeling normalized to that of 3PG in individual patients (mean ± SEM, n = 2 biopsy samples per patient). This ratio serves as an estimate of de novo serine production; purple bars indicate patients designated as having “high” de novo serine production. e) PHGDH protein expression (as determined by RPPA analysis) correlates with de novo serine production (mean, n = 2 biopsy samples per patient). Purple dots represent same patients as purple bars in d). f) Fractional carbon tumor serine labeling normalized to that of 3PG in indicated cancers (mean ± SEM, each point represents a single tumor sample of several collected from each of 5 (brain tumors), 5 (RCC) or 12 (TNBC) patients; one-way ANOVA w/Tukey’s correction). 3PG, 3-phosphoglycerate; PHGDH, phosphoglycerate dehydrogenase; SHMT, serine hydroxymethyl transferase * = p < 0.05, *** = p < 0.001. See also Figure S3.

Figure 5.. Local lactate fermentation from glucose in TNBCs

a) Schematic showing the potential for glucose to label tumor pyruvate/label either via tumor glycolysis or via circulating lactate. Blue circles represent 13C. Glucose, pyruvate, and lactate can exchange between the systemic circulation and tumor. b) Fractional carbon labeling of tumor glucose, pyruvate and lactate relative to the same metabolites in serum at time of biopsy (mean ± SEM, n = 2 biopsies per patient, 3 technical replicates for serum). Patient 17 data are missing due to insufficient serum collection. c) Fractional carbon labeling of glucose, pyruvate and lactate in serum and tumor at time of biopsy (mean ± SEM, n = 11 patients, 3 technical replicates per patient for serum and 2 biopsy samples per patient for tumor, Student’s t-test). d) Fractional carbon labeling of tumor lactate relative to that of the circulation in indicated cancers (mean ± SEM, each point represents a single tumor sample of several collected from each of 30 (NSCLC), 4 (RCC) or 11 (TNBC) patients, one-way ANOVA w/ Tukey’s correction). TNBC patient 17 data are missing due to insufficient serum collection; data for one RCC patient are missing due to poor plasma detection of labeled lactate. Glc, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; 3PG, 3-phosphoglycerate; GLUT, glucose transporter; LDH, lactate dehydrogenase; MCT, monocarboxylic acid transporter. * = p < 0.05, **** = p < 0.0001. See also Figures S4,S5.

Figure 6.. Glucose carbon makes TCA intermediates and associated amino acids in TNBC

a) Pathways from circulating [1,2-13C]glucose to M+2 TCA intermediates and associated amino acids. Blue circles represent 13C. Glucose, pyruvate, lactate, glutamine, glutamate, proline and asparagine can exchange between the systemic circulation and tumor. b) Fractional carbon labeling of tumor succinate, malate, and α-ketoglutarate (αKG) to that of tumor lactate (mean ± SEM, n = 2 biopsies per patient). c) Fractional carbon labeling of tumor malate to that of tumor lactate in the indicated cancers (mean ± SEM, each point represents a single tumor sample of several collected from each of 5 (brain), 30 (NSCLC), 5 (RCC) or 12 (TNBC) patients, one-way ANOVA w/ Tukey’s correction). d) Fractional carbon labeling of indicated tumor amino acid relative to that of respective metabolic precursor in TNBC (mean ± SEM, n = 12 patients, 2 technical replicates per patient). e) Fractional carbon labeling of tumor glutamine relative to that of tumor glutamate in indicated cancers (mean ± SEM, each point represents a single tumor sample of several collected from each of 5 (brain), 30 (NSCLC), 5 (RCC) or 12 (TNBC) patients, one-way ANOVA w/ Tukey’s correction). Three fragments (two from NSCLC, one from RCC) where ratios exceeded 1 were removed as outliers. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. See also Figure S6.

Figure 7.. Evidence for local lactate production through a subset of tumor cells

a) Model depicting sources of tumor lactate and associated equations for calculating the direct contribution of circulating glucose to tumor lactate, reflecting lactate made locally from glucose (α). b) Direct contribution of circulating glucose to tumor lactate (α) in TNBC (mean ± SEM, n = 2 biopsy samples per patient, 3 technical replicates for serum). c) Direct contribution of circulating glucose to tumor lactate (α) in the indicated cancers (mean ± SEM, each point represents a single tumor sample of several collected from each of 5 (brain), 30 (NSCLC), 5 (RCC) or 11 (TNBC) patients, one-way ANOVA w/ Tukey’s correction). d) Lactate/3PG labeling ratio in the indicated cancers (mean ± SEM, each point represents a single tumor sample of several collected from each of 5 (brain), 30 (NSCLC), 5 (RCC) or 12 (TNBC) patients). In line with published work, the lactate/3PG labeling ratio is calculated using the highest labeled forms of lactate and 3PG observed in each study (M+3 in NSCLC, brain tumors, and RCC; M+2 in TNBC). e) Schematic depicting proposed compartmentation of tumor glycolysis. See also Figure S7. ** = p